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Deforestation is a common occurrence driven by agricultural expansion, urbanization, and infrastructure development. These activities often lead to increased human interaction with ecosystems, potentially exposing individuals to Paracoccidioides spores (<i>P. brasiliensis</i> and <i>P. lutzii</i>) found in the soil, resulting in Paracoccidioidomycosis (PCM). This fungal infection is endemic to specific regions in Latin America, such as Brazil, Colombia, Venezuela, and Argentina. Diagnosis typically involves a combination of clinical assessment, imaging techniques, and laboratory examinations. <i>P. lutzii</i> lacks the glycoprotein Gp43, a key antigenic protein utilized in serological tests for PCM diagnosis. In this study, a colorimetric test employing gold nanoparticles (AuNPs) and label-free methodology was employed for <i>P. lutzii</i> detection. The effectiveness of the label-free colorimetric test was assessed using a total of 100 samples. This detection was achieved through the amplification of the gp43 gene and the use of a specific probe (5′CAGGGGTGCG3′) in conjunction with AuNPs. The receiver operating characteristic curve was employed to assess the test, revealing that the method can accurately detect <i>P. lutzii</i> with a sensitivity of 100% and a specificity of 100%. The findings indicate a substantial impact on remote endemic regions attributable to the implementation of cost-effective diagnostic methodologies.